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Transformation
Overview Transformation is the process of inserting a plasmid DNA sequence into a cell of choice through a means of making the cell membrane temporarily permeable. This process can be conducted in both eukaryotic and prokaryotic organisms, but is most commonly used in'' E. coli'' as a means of preparing cells for recombinant expression of a non-native protein or isolation of the same DNA in larger quantities. Transformation is a process that is necessary in streamlining both the industrial and research divisions of science, given that it allows for a much more efficient means of obtaining the material in question through specific amplification or expression inside of a separate organism. Process ] The transformation process starts out with two important materials: competent cells and a DNA plasmid of ]choice. Competent cells are those in which the membrane has been permeabilized through chemical metho ]ds, allowing for exogenous DNA to be taken in. This is typically done through growing up a large amount of cells, collecting them through centrifugation, and placing them into a solution that contains a metal ion (such as magnesium in the chloride salt), a compound that increases membrane permeability (such as dimethyl sulfoxide), and some form of a cryoprotectant for storage (such as polyethylene glycol). These cells have been pre-grown to the early exponential phase of growth as it is believed that this is when they are naturally the most competent in addition to the artificial competence that is going to be induced upon the transformation. C. Chung, S. Niemela, R. Miller. (April, 1989) One-step preparation of competent Escherichia coli: Transformation. Proc. Natl. Acad. Sci. Vol. 86, pp. 2172-2175 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC286873/ PubMed] The second important material is a DNA plasmid that has been modified, typically using restriction endonuclease ligation, to contain the specific gene of interest that had previously been amplified using PCR. This plasmid typically contains a means of selection, classically antibiotic resistance, as a post-transformation test to separate cells that have taken in the plasmid and those that have not. Plasmids are then introduced into the DNA through a heat-shocking process in which the competent cells and plasmid are incubated together at a cold temperature, introduced to a high temperature water bath (typically around 40C) for a short period of time to allow the added ions to disrupt the plasma membrane of the cells, and then put back on ice to re-close the membrane with the exogenous DNA now inside the cells. These cells are then grown in a separate incubation in fresh media and plated on selective plates. The colonies that grow at the end of this incubation will be cells that have taken up the specific plasmid, as those cells that have taken up the plasmid will have the ability to survive on the antibacterial plates.A. Froger and J. Hall. (2007) Transformation of Plasmid DNA into E. coli Using the Heat Shock Method. J Vis Exp. (6): 253. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557105/?report=classic PubMed] Transformation is not limited to heat shocking procedures. Another popular method, utilizing the same basic concept, uses a process called electroporation to permeabilize the cell membranes. In this system, instead of utilizing a chemical to disrupt the plasma membrane, the cells are subjected to an electrical pulse that causes the membrane to develop holes in which the plasmid DNA can then enter. Following the pulse, the cells are then transferred to growth medium and the protocol continues as the normal chemical method does. This method has been shown to have an extremely high level of efficiency in comparison to the normal chemical method.W. Dower, J. Miller, C. Ragsdale. (July 1988) Nucleic Acids Res. 16(13): 6127–6145. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC336852/ PubMed] Uses ] Transformation protocols have been developed for bacterial, yeast, plant, and animal cell (known as transfection) types and are commonly used by researchers for both DNA purification and recombinant protein expression. Methods for protein purification generally involve a means of amplifying expression by means of utilizing the Lac operon in the plasmid, induced with Isopropyl β-D-1-thiogalactopyranoside, to force the cells to express high amounts of protein which can be then purified from the cells post-incubation and lysis. This method typically involves a type of column chromatography or ammonium sulfate fractionation.A. Marbach, K. Bettenbrock. (January 2012) lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. J Biotechnol. 157(1):82-8 [http://www.ncbi.nlm.nih.gov/pubmed/22079752 PubMed] In terms of DNA purification, cells are typically grown in roughly 2-3 mL of media after transformation and selection and subjected to a prep system that lyses cells and separates out DNA through the use of a microfuge column system.QIAprep Miniprep Handbook [http://public.wsu.edu/~kahn_sci/Flow/E2-QIAprep_Miniprep_Handbook.pdf Source] References